Lyme disease is an important public health problem in the US. This emerging disease is underdiagnosed, and the late stages of Lyme disease are difficult to treat. The causative agent Borrelia burgdorferi (Bb) is maintained in a complex enzootic cycle involving its arthropod vector (Ixodes ticks) and a rodent mammalian host. Bb must dramatic alters its surface proteins in order to adapt to two markedly different host environments during tick feeding. Previously, we and others have established that the RpoN-RpoS pathway functions as a major mechanism underlying differential gene expression during the spirochete's enzootic cycle. The goal of this application is to fill in the major gap in the understanding of this essential pathway, that is, how multiple signals and regulators coordinately activate / regulate this pathway. Our preliminary findings support the following hypotheses, which will be tested in three Specific Aims: 1) This pathway connects Bb metabolism and virulence. 2) Acetylation plays an important role in the activation of this pathway and in the enzootic cycle of Bb. 3) the second messenger c-di-GMP modulates this pathway via a new c-di-GMP effector. This is a collaborative project including four experienced scientists with expertise in Bb gene regulation (Yang; PI), bacterial acetylation (Wolfe), Bb physiology (Gherardini), and c-di-GMP (Gomelsky). Outcomes of the work will reveal how Bb has evolved to connect metabolism and virulence and how acetylation modulates differential gene expression during the enzootic cycle of Bb. This work will also reveal a novel role of c-di-GMP in host adaptation of vector-borne pathogens. Our proposal will fill a major gap in our understanding of how Bb adapts to both arthropod and mammalian hosts. Outcomes of this work also will set a foundation for developing strategies to block the enzootic cycle of Bb, thus limiting its ability to cause human disease.